1. Field of the Invention
The present invention concerns the removal of process chemicals from labile biological mixtures by hydrophobic interaction chromatography. More particularly, the present invention concerns the removal of lipid soluble process chemicals, for example, chemicals used in the inactivation of viruses in plasma, cryoprecipitates and plasma-derived therapeutic preparations.
2. Background Information
Blood can contain each of several different viruses including but not limited to hepatitis B virus (HBV), non-A, non-B hepatitis virus (NANBHV), cytomegalovirus, and immunodeficiency viruses. It is highly desirable to inactivate these viruses in the course of preparing blood products and prior to the therapeutic application of blood and blood fractions. Both physical (e.g., heat, irradiation) and chemical (e.g., aldehydes, organic solvents, detergents, etc.) methods have been used to inactivate viruses such as HBV in mammalian blood and blood fractions. Inactivation renders a virus non-infectious and non-pathogenic.
Numerous attempts have been made to inactivate viruses such as hepatitis B virus (HBV) in mammalian, especially human, blood plasma. It is the practice in some countries to effect inactivation of the hepatitis B virus in the blood plasma by bringing the plasma into contact with a viral inactivating agent which crosslinks the proteinaceous portions of hepatitis B virus or which interacts with the nucleic acid of the virus. For instance, it is known that hepatitis B virus is inactivated by contact with an aldehyde, such as formaldehyde.
Among the procedures for inactivating viruses are the use of lipid solvents with the addition of surface active agents (A. M. Prince, B. Horowitz, B. Brotman et al, "Inactivation of Hepatitis B and Hutchinson Strain non-A, non-B Hepatitis Viruses by Exposure to Tween 80 and Ether", Vox Sang, (1984 ), 46, 36-43; A. M. Prince, B. Horowitz and B. Brotman, "Sterilisation of Hepatitis and HTLV-III Viruses By Exposure to Tri(n-Butyl)Phosphate and Sodium Cholate", The Lancet, 706-710, Mar. 29, 1986), and lipid solvents without the additive of surface active agents (S. M. Feinstone, K. B. Mihalik, T. Kamimura et al, "Inactivation of Hepatitis B Virus and non-A, non-B Hepatitis by Chloroform, Infect. Immunol., (1983), 41, 816-821; D. W. Bradley, J. E. Maynard, H. Popper et al, "Posttransfusion non-A, non-B Hepatitis: Physiochemical Properties of Two Distinct Agents", J. Infect. Dis., (1983), 148, 254-265).
U.S. Pat. Nos. 4,481,189 and 4,540,573, the entire contents of which are incorporated by reference herein, describe the use of organic solvent/detergent pairs to reduce the infectivity of hepatitis viruses and certain other viruses contained in or added to plasma and plasma products by several orders of magnitude. An example of such a pair is tri-n-butyl phosphate, an organic solvent, and TRITON X-100, a non-ionic detergent.
Solvent/detergent treatment under appropriate conditions of temperature and contact time effectively disassembles viruses that have envelope proteins associated with lipid, while having negligible effect on the molecular conformations and biological activities of sensitive blood plasma proteins. The independent effects of organic solvents and detergents in disassembling and attenuating viruses can be facilitated by the presence of both. Merocyanine, beta-propiolactone and cis-platin are among other agents that are applied to blood to inactive viruses, though by mechanisms other than envelope disruption.
Removal of organic solvents, detergents and other virus-inactivating agents from biological products is necessary if a particular substance is not well tolerated by humans or other biological systems in which it is to be used, e.g., in tissue cultures. In the preparation of purified plasma proteins such as coagulation factor VIII or mixtures of selected porteins, the separation of the desired product from the virus-inactivating agents is often facilitated by a purification process. Thus, precipitation of the desired protein or positive adsorption by an immobilized product-specific ligand can often reduce the level of residual agents to "tolerable" levels by allowing the majority to be washed away.
Other methods used to achieve removal of lipid/detergent micelles from membrans-protein complexes may be applicable to removal of the same from plasma products and other biologic products. These have been based on differences in size, buoyant density, charge, binding affinity, phase partitioning and solvent partitioning (A. Helenius and K. Sinous, "Solubilization of Membranes by Deterents", Biochem. Biophys. ACTA, (1975), 415,29-79).
In the instance of whole blood plasma, blood serum, cryodepleted plasma or cryoprecipitate for direct use in transfusion, implementation of the organic solvent/detergent method of virus sterilization has not occurred. Because preparation of these materials does not involve steps for fractionation, purification, or refinement, there is no convenient opportunity to effect removal of the inactivating agents by the methods suggested above. An efficient removal method is further constrained by the necessity of leaving the composition and biological activity of the preparation substantially intact.
Another difficulty in preparing virus sterilized plasma and cryoprecipitate is in filtering the plasma after treatment to maintain bacterial sterility without loss of labile proteins and biological activity.
Thus, because virus sterilization techniques have not been applied to whole blood plasma, etc., virus infectivity upon infusion remains, estimated at 0.05% for hepatitis B and 3% for non-A, non-B hepatitis transmission.
Exogenous chemicals are frequently added to biological mixtues to stimulate synthesis, inactivate viruses contained therein and to stabilize or purify desired components present in the mixture. It is desirable to remove these chemicals without otherwise affecting the structure and function of the desired components. For example, the synthesis of certain desired biological products can be induced or enhanced in cell cultures by introduction of phorbol esters into the culture fluid. For example, mezerein may be used to induce gamma interferon production by cultured leukocytes (Y. K. Yip, R. H. L. Pang, J. O. Oppenheim, M. S. Nashbar, D. Henriksen, T. Zerebeckyj-Eckhardt, J. Vilcek, "Stimulation of Human Gamma Interferon Production by Diterpene Esters", Infect. and Immun., (1981) 131-139) or to augment secretion of tumor necrosis factor by cells that produce it (B. D. Williamson, E. A. Carswell, B. Y. Rubin, J. S. Prendergast, II. J. Old, "Human Tumor Necrosis Factor Produced by Human B-cells Lines: Synergistic Cytoxic Interaction Human Interferon", Proc. Natl. Acad. Sci., U.S.A., (1983), 80, 5397-5401).
Before use in man, phorbol esters must be removed from lymphokine preparations because of the carcinogenic properties of these compounds. Heretofore, phorbol esters have been removed by precipitation, chromatographic, or molecular exclusion processes, (B. Y. Rubin, S. L. Anderson, S. A. Sullivan, B. D. Williamson, E. A. Carswell, L. J. Old, "Purification and Characterization of a Human Tumor Necrosis Factor from the LukII Cell Line", Proc. Natl. Acad. Sci., U.S.A., (1985), 82, 6637-6641).
A method for removal of TNBP and other lipid soluble process chemicals from complex biological mixtures by extraction with vegetable oils, e.g., soy bean oil, is described in U.S. patent application Ser. No. 846,374, filed, Mar. 31, 1986, now U.S. Pat. No. 4,789,545. This method does not remove most detergents.
A similar method for removal of TNBP, detergents and other lipid soluble chemicals by extraction with long-chain alcohols or halogenated esters is described in pending U.S. patent application Ser. No. 07/139,502 filed Dec. 30, 1987, now U.S. Pat. No. 4,909,940. Shortcomings of this method are that expensive and/or noxious chemicals are required and that additional steps must be taken to reduce to tolerable levels any residual extraction agents. An alternative method that efficiently removed TNBP and detergents and did not present these problems would be desirable.
Hydrophobic interaction chromatography (HIC) is a commonly used tool in the biochemists' arsenal of molecular separation techniques. A description of the principles of HIC is given in S. Hjerten, "Some General Aspects of Hydrophobic Interaction Chromatography", J. of Chromatography, (1973), 87, 325-331. Briefly, a lipid-like moiety such as an alkyl chain coupled to an insert matrix is used to partition molecules containing similar hydrophobic domains from aqueous solutions by virtue of their mutual affinity. The alkyl chain may range from two to twenty-four or more carbons in length and may be linear or branched and may contain or terminate in other hydrophobic groups such as a phenyl ring. Increasing chain length results in media with greater hydrophobic character.
In practice, the strength of the hydrophobic interactions is also influenced by the ionic strength, pH and polarity of the solvent. For example, a high concentration (i.e., 4 molar) of ammonium sulphate in the solvent would promote hydrophobic interaction and, hence, binding, between the resin and the hydrophobic domains of the solute proteins. Following absorption, the proteins can be eluted from the resin by using a buffer with lower ionic strength, chaotropic ions, and/or polarity-lowering additives, such as ethylene glycol or detergents. In addition to resins that react strictly through hydrophobicity, there are materials that work via a combination of hydrophobic and ionic interactions such as amino-hexlyl Sepharose (LKB/Pharmacia, Piscataway, N.J.) which incorporates an amino group at the end of a six carbon alkyl chain as the active function. Among the many plasma proteins that have been purified by these methods are albumin, transferrin, thyroglobulin, lactic dehydrogenase, beta lipoproteins, coagulation factors and immunoglobulins.
The inert matrix to which the hydrophobic groups are bound in the preparation of HIC media may be comprised of a polysaccharide, such as agarose or silica or other polymers. Agarose based media are relatively soft and, in a typical chromatography system, require slow flow rates and result in lengthy separation procedures. Silica based media, being incompressible, are typically employed in high pressure chromatography systems, but may be used in low pressure systems at relatively high flow rates.
Though generally used for the isolation of proteins, hormones, and other biological molecules from complex biological mixtures, HIC has also been used to remove detergent from certain biological preparations where the detergent was used to dissociate membranes. For example, C18 HIC medium in a cartridge (Sep-Pak, Waters Associates, Milford, Mass.), as well as an ion exchange column was used to remove TRITON X-100, protein, salts and other interfering compounds from rat brain homogenates in order to facilitate the measurement of inositol in the tissue extract (S. E. Laursen, H. R. Knull and J. K. Belknap, "Sample Preparation for Inositol Measurement: Sep-Pak C18 Use in Detergent Removal", Analytical Biochemistry, 153, 387-390 (1986)).